Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes when exposed to low oxygen tension. Polymerization makes the red blood cells rigid so that they cannot traverse some blood vessels leading to blockage of these vessels which then leads to significant morbidity and mortality. Nitric Oxide (NO) is currently being tested as a treatment for sickle cell disease due to (among other things) its role as a vasodilator. NO is synthesized in endothelial cells of blood vessels and diffuses to neighboring smooth muscle cells where it acts as a signaling molecule, causing muscle relaxation and vasodilation. Sickle red blood cells are also fragile, rupturing during transit in the circulation. For many years, this hemolytic anemia aspect of sickle cell disease was not viewed as a playing a major role in the pathophysiology of the disease. However, several groups have now begun to re-examine the consequences of hemolysis and the hypothesis that cell-free hemoglobin that is released efficiently scavenges NO, leading to an NO-related deficiency associated with sickle cell disease. This project aims to elucidate the mechanism of hemolysis in sickle cell disease - how does the hemoglobin mutation lead to increased red blood cell fragility? In addition, the mechanism of reduced NO scavenging by red cell encapsulated hemoglobin (compared to cell-free hemoglobin) will be thoroughly explored. Differences in how NO reacts in sickle cell blood and that from volunteer non-patients will be determined. Finally, we will explore basic scientific aspects of a mechanism of restoring effective NO response in patients using the anion nitrite. The laboratories participating in this project have recently shown that, contrary to the existing paradigm, nitrite acts as a vasodilator in human circulation. In addition, it was shown that this action of nitrite may be due to a novel allosterically-controlled function of hemoglobin. The study employs a vast array of biophysical techniques including electron paramagnetic resonance spectroscopy, kinetic absorption spectroscopy, laser diffraction, and computational simulations. Techniques have been developed so that studies can be made on whole blood, so that physiologically relevant conditions can be assessed.